MAMMALIAN EXPRESSION VECTOR pUHAB

ABSTRACT

The present invention relates to the construction and utilization of a new mammalian expression vector that contains a unique multiple cloning site (MCS), designated pUHAB. The pUHAB vector comprises a high copy replication origin (ColE1), a drug resistance gene (TK-Hygromycin), and a human cytomegalovirus promoter operably associated with a unique intron (hCMV/intron). Further, pUHAB comprises a selectable marker conferring resistance to kanamycin in bacterial cells, and a phage f1(+) region. pUHAB can be used to transiently or stably express cloned genes when transfected into mammalian cells. The invention also encompasses kits and host cells and cell lines comprising pUHAB, and methods of producing a recombinant protein using pUHAB.

This application claims the benefit of U.S. provisional patentapplication no. 61/113,824; filed Nov. 12, 2008; which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the construction and utilization of anew mammalian expression vector that contains a unique multiple cloningsite (MCS), designated pUHAB. The pUHAB vector comprises a high copyreplication origin (ColE1), a drug resistance gene (TK-Hygromycin), anda human cytomegalovirus promoter operably associated with a uniqueintron (hCMV/intron). Further, pUHAB comprises a selectable markerconferring resistance to kanamycin in bacterial cells, and a phage f1(+)region. pUHAB can be used to transiently or stably express cloned geneswhen transfected into mammalian cells. The invention also encompasseskits and host cells and cell lines comprising pUHAB, and methods ofproducing a recombinant protein using pUHAB.

BACKGROUND OF THE INVENTION

Culturing cells for the commercial production of therapeutic proteins isa costly process. The equipment required is expensive and research anddevelopment and production costs are high. Development of cell cultureprocesses which maximize the quantity of therapeutic protein producedper liter of cell culture will minimize the resources necessary toproduce a given quantity of the protein. It is, thus, desirable to usecommercially viable reagents which produce large quantities of proteins.

Many naturally occurring cells do not produce large quantities ofdesired proteins, under standard culture conditions. Rather, extensiveresearch and development of cell culture processes, which coax cells inculture to generate large quantities of therapeutic protein, must beperformed. Typically, identifying plasmid vectors useful for expressinga protein at a high level requires a significant amount of inventiveinput.

SUMMARY OF THE INVENTION

The present invention provides, in part, a new expression vector. In oneembodiment, the invention provides a vector characterized by a long andunique multiple cloning site. The multiple cloning site may comprise 10,11, 12, 13, 14 or 15 restriction sites selected from the groupconsisting of: AflII, HpaI, AvrII, EcoRV, Acc651, KpnI, PacI, NotI,BstZ17I, SrfI, ApaI, NheI, BglII, SphI and BamHI. In one embodiment, themultiple cloning site comprises all of said restriction sites, e.g., inthe order:5′-AflII-HpaI-AvrII-EcoRV-Acc651-KpnI-PacI-NotI-BstZ17I-SrfI-ApaI-NheI-BglII-SphI-BamHI-3′.In an exemplary embodiment, the multiple cloning site comprises thenucleotide sequence set forth in SEQ ID NO: 2.

The vector may further comprise a promoter located upstream of or withinthe multiple cloning site. In one embodiment, the promoter is the humancytomegalovirus (hCMV) promoter. The promoter may be operably associatedwith an intron that enhances expression from the promoter. In oneembodiment, the nucleotide sequence of the intron is comprised by thenucleotide sequence of SEQ ID NO: 1.

The vector may further comprise at least 1, 2, 3 or 4 elements selectedfrom the group consisting of: a selectable marker for eukaryotic cells(e.g., a TK-Hygromycin gene); a prokaryotic origin of replication (e.g.,a ColE1 origin of replication); a bacterial drug resistance marker(e.g., a kanamycin resistance gene); and a phage f1 region (e.g., aphage f1(+) region). In one embodiment, the vector comprises all ofthese elements. The vector may further comprise a terminator/polyAaddition site.

The invention also provides a vector comprising an hCMV promoteroperably associated with an intron that enhances expression from saidpromoter, wherein the nucleotide sequence of the intron is comprised bythe nucleotide sequence of SEQ ID NO: 1. The vector may further compriseat least 1, 2, 3, 4 or 5 elements selected from the group consisting of:a multiple cloning site (e.g., the multiple cloning site of SEQ ID NO:2); a selectable marker for eukaryotic cells (e.g., a TK-Hygromycingene); a prokaryotic origin of replication (e.g., a ColE1 origin ofreplication); a bacterial drug resistance marker (e.g., a kanamycinresistance gene); and a phage f1 region (e.g., a phage f1(+) region). Inone embodiment, the vector comprises all of these elements. The vectormay further comprise a terminator/polyA addition site.

In an exemplary embodiment, the vector comprises the nucleotide sequenceset forth in SEQ ID NO: 1.

The vector of the invention may be an expression vector for use inexpression of a recombinant polypeptide in a mammalian host cell ororganism. In this embodiment, the vector comprises a heterologous DNAsequence encoding the recombinant polypeptide, wherein the heterologousDNA sequence is operably linked to the promoter sequence.

The invention also provides a host cell comprising the vector of theinvention. Also encompassed is a method for producing a recombinantpolypeptide in a mammalian host cell, comprising introducing the vectorof the invention into the host cell under conditions which allow forexpression of the polypeptide. The recombinant polypeptide may then bepurified.

The invention also provides a kit comprising the vector of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Nucleotide sequence of pUHAB (SEQ ID NO: 1).

FIG. 2. Plasmid map of pUHAB.

Feature Map Element Start End Beta Globin polyA signal 5 243TK-Hygromycin 289 2298 f1(+) origin 2467 2923 KN(R) (Tn903 type I) 35234339 ColE1 origin 4884 5824 hCMV/intron 6084 6925 MCS 6926 5

FIG. 3. Nucleotide sequence of Vector A (SEQ ID NO: 3).

FIG. 4. Plasmid map of Vector A.

Feature Map Element Start End hCMV/intron 5 846 (complementary) ColE1origin 1106 2046 (complementary) KN(R) (Tn903 type I) 2591 3407(complementary) f1(+) origin 4007 4463 (complementary) TK-Hygromycin4632 6641 Beta Globin polyA signal 6687 6925 (complementary) IgG1constant 6950 7930 (complementary) VDJ (variable region) 7942 8391(complementary)

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an expression vector useful forrecombinant protein expression in any cell, for example in a mammaliancell, a bacterial cell, a yeast cell or an insect cell. The vector maybe used to transiently or stably express a broad range of recombinantproteins. The multiple cloning site of the vector offers many common andrare restriction sites to accommodate a variety of expression cassettes.

The present invention includes a vector comprising or consisting of thenucleotide sequence of SEQ ID NO: 1.

Molecular Biology

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explained inthe literature. See, e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, Second Edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook, et al.,1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N.Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985));Transcription And Translation (B. D. Hames & S. J. Higgins, eds.(1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); ImmobilizedCells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide ToMolecular Cloning (1984); F. M. Ausubel, et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

A “polynucleotide,” “nucleic acid” or “nucleic acid molecule” includesthe polymeric form of ribonucleosides (adenosine, guanosine, uridine orcytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”).Polynucleotides of the invention can be in any form, including circular,linear, double-stranded or single-stranded.

A “polynucleotide sequence,” “nucleic acid sequence” or “nucleotidesequence” is a series of nucleotides in a nucleic acid, such as DNA orRNA, and means any chain of two or more nucleotides.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA or peptide, is a nucleotide sequence that, when expressed,results in production of the product.

The term “gene” means a DNA sequence that codes for or corresponds to aparticular sequence of ribonucleotides or amino acids which comprise allor part of one or more RNA molecules or proteins, and may or may not beoperably linked to regulatory DNA sequences, such as promoter sequences,which determine, for example, the conditions under which the gene isexpressed. Genes may be transcribed from DNA to RNA which may or may notbe translated into an amino acid sequence.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of no more than about 300 nucleotides (e.g., 30, 40, 50, 60,70, 80, 90, 150, 175, 200, 250 or 300), that may be hybridizable to agenomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding agene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotidesare usually single-stranded, but may be double-stranded.Oligonucleotides can be labeled, e.g., by incorporation of³²P-nucleotides, ³H-nucleotides, ¹⁴C-nucleotides, ³⁵S-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. In one embodiment, a labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. In another embodiment,oligonucleotides (one or both of which may be labeled) can be used asPCR primers, either for cloning full length or a fragment of the gene,or to detect the presence of nucleic acids. Generally, oligonucleotidesare prepared synthetically, e.g., on a nucleic acid synthesizer.

A “protein sequence,” “peptide sequence” or “polypeptide sequence,” or“amino acid sequence” refers to a series of two or more amino acids in aprotein, peptide or polypeptide.

“Protein,” “peptide” or “polypeptide” includes a contiguous string oftwo or more amino acids.

The term “isolated polynucleotide” or “isolated polypeptide” includes apolynucleotide (e.g., RNA or DNA molecule, or a mixed polymer) or apolypeptide, respectively, which is partially or fully separated fromother components that are normally found in cells or in recombinant DNAexpression systems or any other contaminant. These components include,but are not limited to, cell membranes, cell walls, ribosomes,polymerases, serum components and extraneous genomic sequences.

An isolated polynucleotide or polypeptide will, preferably, be anessentially homogeneous composition of molecules but may contain someheterogeneity.

As used herein, the term “functional variant” refers to a variantnucleotide or polypeptide that produces substantially the samebiological effect as the original nucleotide or polypeptide.

Variants included in the invention may contain individual substitutions,deletions or additions to the original nucleic acid or polypeptidesequences. Such changes will alter, add or delete a single amino acid ora small percentage of amino acids (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9% or 10%) in the encoded sequence. Variants are referred to as“conservatively modified variants” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.

“PCR amplification” of DNA as used herein includes the use of polymerasechain reaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki et al., Science (1988) 239:487.

The term “host cell” includes any cell of any organism that is selected,modified, transfected, transformed, grown, or used or manipulated in anyway, for the production of a substance by the cell, for example theexpression or replication, by the cell, of a gene, a DNA or RNA or aprotein. For example, a host cell may be a mammalian cell, a bacterialcell, a yeast cell or an insect cell.

A further aspect of the present invention relates to a host cell or hostcell line comprising the vector of the invention. In one embodiment, thehost cell is a mammalian cell. Examples of mammalian host cells include,by way of nonlimiting example, Chinese hamster ovary (CHO) cells, CHO-K1cells, CHO-DXB-11 cells, CHO-DG44 cells, bovine mammary epithelialcells, mouse Sertoli cells, canine kidney cells, buffalo rat livercells, human lung cells, mouse mammary tumor cells, rat fibroblasts,bovine kidney (MDBK) cells, NSO cells, SP2 cells, TRI cells, MRC 5cells, FS4 cells, HEK-293T cells, NIH-3T3 cells, HeLa cells, babyhamster kidney (BHK) cells, African green monkey kidney (COS) cells,human hepatocellular carcinoma (e.g., Hep G2) cells, A549 cells, etc. Inone embodiment, the mammalian host cell is a human host cell. Mammalianhost cells can be cultured according to methods known in the art (see,e.g., J. Immunol. Methods 56:221 (1983), Animal Cell Culture: APractical Approach 2nd Ed Rickwood, D. and Hames, B. D., eds. OxfordUniversity Press, New York (1992)).

Vectors of the invention can also be introduced into a bacterial cell.In one embodiment, competent E. coli are transformed. Examples ofsuitable E. coli include DH1, DH5, DH5α, XL1-Blue, SURE, SCS110, OneShotTop 10, and HB101.

Vectors of the invention may be introduced into host cells according toany of the many techniques known in the art, e.g., dextran-mediatedtransfection, polybrene-mediated transfection, protoplast fusion,electroporation, calcium phosphate co-precipitation, lipofection, directmicroinjection of the vector into nuclei, or any other means appropriatefor a given host cell type.

A “cassette” or an “expression cassette” refers to a DNA coding sequenceor segment of DNA that codes for an expression product (e.g., peptide orRNA) that can be inserted into a vector, e.g., at defined restrictionsites. The expression cassette may comprise a promoter and/or aterminator and/or polyA signal operably linked to the DNA codingsequence.

The sequence of a nucleic acid may be determined by any method known inthe art (e.g., chemical sequencing or enzymatic sequencing). “Chemicalsequencing” of DNA includes methods such as that of Maxam and Gilbert(Proc. Natl. Acad. Sci. USA (1977) 74:560), in which DNA is randomlycleaved using individual base-specific reactions. “Enzymatic sequencing”of DNA includes methods such as that of Sanger (Sanger et al., Proc.Natl. Acad. Sci. USA (1977) 74:5463).

In general, a “promoter” or “promoter sequence” is a DNA regulatoryregion capable of binding an RNA polymerase in a cell (e.g., directly orthrough other promoter-bound proteins or substances) and initiatingtranscription of a coding sequence. A promoter sequence is, in general,bounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at any level. Within thepromoter sequence may be found a transcription initiation site(conveniently defined, for example, by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. The promoter may be operably associatedwith or operably linked to other expression control sequences, includingenhancer and repressor sequences or with a nucleic acid to be expressed.An expression control sequence is operably associated with or operablylinked to a promoter if it regulates expression from said promoter.

Promoters which may be used to control gene expression include, but arenot limited to, SRα promoter (Takebe et al., Molec. and Cell. Bio.8:466-472 (1988)), the human CMV immediate early promoter (Boshart etal., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)),the mouse CMV immediate early promoter, the SV40 early promoter region(Benoist at al., Nature 290:304-310 (1981)), the Orgyia pseudotsugataimmediate early promoter, the herpes thymidine kinase promoter (Wagnerat al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatorysequences of the metallothionein gene (Brinster et al., Nature 296:39-42(1982)); prokaryotic expression vectors such as the β-lactamase promoter(Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)),or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25(1983)); and promoter elements from yeast or other fungi such as theGAL1, GAL4 or GAL10 promoter, the ADH (alcohol dehydrogenase) promoter,PGK (phosphoglycerol kinase) promoter or the alkaline phosphatasepromoter.

Viral long terminal repeat promoters such as the mouse mammary tumorvirus long terminal repeat (MMTV-LTR) (Fasel et al, EMBO J. 1(1):3-7(1982)), the moloney murine sarcoma virus long terminal repeat (Reddy etal., Proc. Natl. Acad. Sci. USA 77(9): 5234-5238 (1980)), the moloneymurine leukemia virus long terminal repeat (Van Beveren at al., Proc.Natl. Acad. Sci. USA 77(6): 3307-3311 (1980)), the HIV LTR (GenbankAccession No. AB100245), the bovine foamy virus LTR (Genbank AccessionNo. NC_(—)001831), RSV 5′-LTR (Genbank Accession No. K00087), the HIV-2LTR (Genbank Accession No. NC_(—)001722), an avian retroviral LTR (Ju atal., Cell 22: 379-386 (1980)) and the human herpesvirus LTR (GenbankAccession No. NC_(—)001806) may be included in the vectors of thepresent invention.

Other acceptable promoters include the human CMV promoter, the humanCMV5 promoter, the murine CMV promoter, the EF1α promoter, the SV40promoter, a hybrid CMV promoter for liver specific expression (e.g.,made by conjugating CMV immediate early promoter with thetranscriptional promoter elements of either human α1-antitrypsin (HAT)or albumin (HAL) promoter), or promoters for hepatoma specificexpression (e.g., wherein the transcriptional promoter elements ofeither human albumin (HAL; about 1000 bp) or human α1-antitrypsin (HAT,about 2000 bp) are combined with a 145 by long enhancer element of humanα1-microglobulin and bikunin precursor gene (AMBP); HAL-AMBP andHAT-AMBP).

In addition, bacterial promoters, such as the T7 RNA Polymerase promoteror the tac promoter, may be used to control expression.

In one embodiment, the promoter is the human CMV (hCMV) promoter. ThehCMV promoter provides a high level of expression in a variety ofmammalian cell types.

A coding sequence is “under the control of”, “functionally associatedwith”, “operably linked to” or “operably associated with”transcriptional and translational control sequences in a cell when thesequences direct or regulate expression of the sequence. For example, apromoter operably linked to a gene will direct RNA polymerase mediatedtranscription of the coding sequence into RNA, preferably mRNA, whichmay then be spliced (if it contains introns) and, optionally, translatedinto a protein encoded by the coding sequence. A terminator/polyA signaloperably linked to a gene terminates transcription of the gene into RNAand directs addition of a polyA signal onto the RNA.

The terms “express” and “expression” mean allowing or causing theinformation in a gene, RNA or DNA sequence to become manifest; forexample, producing a protein by activating the cellular functionsinvolved in transcription and translation of a corresponding gene.“Express” and “expression” include transcription of DNA to RNA and ofRNA to protein. A DNA sequence is expressed in or by a cell to form an“expression product” such as an RNA (e.g., mRNA) or a protein. Theexpression product itself may also be said to be “expressed” by thecell.

The term “transformation” means the introduction of a nucleic acid intoa cell. The introduced gene or sequence may be called a “clone”. A hostcell that receives the introduced DNA or RNA has been “transformed” andis a “transformant” or a “clone.” The DNA or RNA introduced to a hostcell can come from any source, including cells of the same genus orspecies as the host cell, or from cells of a different genus or species.Examples of transformation methods which are very well known in the artinclude liposome delivery, electroporation, CaPO₄ transformation,DEAE-Dextran transformation, microinjection and viral infection.

The present invention includes vectors which comprise polynucleotides ofthe invention. The term “vector” may refer to a vehicle (e.g., aplasmid) by which a DNA or RNA sequence can be introduced into a hostcell, so as to transform the host and, optionally, promote expressionand/or replication of the introduced sequence.

The polynucleotides of the invention may be expressed in an expressionsystem. The term “expression system” means a host cell and compatiblevector which, under suitable conditions, can express a protein ornucleic acid which is carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells and baculovirus vectors, and mammalian hostcells and vectors such as plasmids, cosmids, BACs, YACs and viruses suchas adenovirus and adenovirus associated virus (AAV).

Vectors

The invention provides a new expression vector. In one embodiment, thevector comprises a unique and long multiple cloning site that providesthe vector with the flexibility to incorporate a variety of recombinantgenes in complex arrangements for expression in a mammalian cell ororganism. In one embodiment, the vector comprises a multiple cloningsite comprising restriction sites selected from the group consisting of:AflII, HpaI, AvrII, EcoRV, Acc651, KpnI, PacI, NotI, BstZ17I, SrfI,ApaI, NheI, BglII, SphI, and BamHI. The multiple cloning site maycomprise 10, 11, 12, 13, 14 or 15 of said restriction sites. Themultiple cloning site may comprise, for example, the nucleotide sequenceof SEQ ID NO: 2 as shown below:

(SEQ ID NO: 2)                                           KpnI AfIII    HpaI        AvrII    EcoRV     Acc651 --------------     -------   -------   ------- CTTAAGAGTG TTAACGCGAC CTAGGTAAGATATCCTTGGT ACCGTG PacI         NotI      BstZ17I     SrfI       ApaI-------   ---------    -------   ---------   ----- TTAATTAACTGGCGGCCGCTGTGT ATACGTGGCC CGGGCTGGGG GC      NheI      BgIII     SphI      BamHI-    -------   -------   -------   ------ CCATAGCT AGCGTTAGATC TCTGGCATGCGCTGGATCC

The present invention contemplates vectors comprising theabove-indicated multiple cloning site in the orientation shown or in theopposite orientation.

In one embodiment of the invention, the vector comprises a promoterwhich is essentially any RNA polymerase-dependent promoter, e.g., an RNApolymerase II-dependent promoter.

In certain embodiments, the vector may comprise a combination of apromoter and an intron that increases gene expression from saidpromoter. The intron may be synthetic or naturally-derived. In oneembodiment, the intron increases gene expression from the hCMV promoter.In an exemplary embodiment, the intron is a unique intron including thenucleotide sequence:

(SEQ ID NO: 4) CA GGTAAGTTTA AAGCTCAGGT CGAGACCGGG CCTTTGTCCG GCGCTCCCTTGGAGCCTACC TAGACTCAGC CGGCTCTCCA CGCTTTGCCT GACCCTGCTT GCTCAACTCTACGTCTTTGT TTCGTTTTCT GTTCCTTTCT CTCCACAGGC.

The combination of the human CMV promoter and the intron (hCMV/intron)provides higher expression levels than those achieved by using hCMValone.

The vector may also include one or more further regulatory elements inaddition to the promoter and the intron, such as enhancer elements,splicing signals, polyadenylation signals, termination signals, RNAexport elements, secretion signals, internal ribosome entry sites, andthe like.

In one embodiment, the vector of the invention comprises a selectivemarker which allows for selection of eukaryotic (e.g., mammalian) hostcells into which the vector has been introduced. The selective markermay, for example, confer resistance to drugs such as G418, hygromycin ormethotrexate. In one embodiment of the invention, the selective markeris a gene providing positive selection for hygromycin resistance in bothprokaryotes and eukaryotes, fused to the thymidine kinase promoter(TK-Hygromycin).

In one embodiment, the vector of the invention comprises a prokaryoticantibiotic resistance marker such as the ampicillin resistance gene orthe kanamycin resistance gene.

In one embodiment, the vector of the invention comprises a phage f1region comprising the origin of replication from the f1 filamentousphage, allowing rescue of single-stranded DNA upon co-infection withhelper phage. This single-stranded DNA may be used for, e.g.,dideoxynucleotide sequencing or site-directed mutagenesis. In oneembodiment, the phage f1 region is a phage f1(+) region.

In certain embodiments of the invention, the vector is a plasmid. Theplasmid vector may comprise a prokaryotic origin of replication to allowautonomous replication within a prokaryotic host cell. In oneembodiment, the prokaryotic origin of replication is the high copy ColE1origin of replication, which allows the vector to produce the highlevels of plasmid DNA required for large scale transient transfections.

In an embodiment of the invention, the vector comprises a multiplecloning site of SEQ ID NO: 2, a ColE1 high copy origin of replication, aTK-Hygromycin drug resistance gene, a human CMV promoter operablyassociated with an intron, a kanamycin resistance marker, and a phagef1(+) region. The invention also encompasses vectors wherein any or allof these elements are replaced by functional variants of said elements.In one embodiment, the vector is described by the plasmid map of FIG. 2.

In one embodiment of the invention, the vector is an expression vectorcapable of expressing a recombinant polypeptide in a host cell ororganism. In an exemplary embodiment, the host cell or organism is amammalian host cell or organism.

The expression vector comprises, in an embodiment of the invention, aterminator sequence to terminate transcription. Further, the expressionvector may comprise a polyadenylation (polyA) signal for stabilizationand processing of the 3′ end of an mRNA transcribed from the promoter.PolyA signals include, for example, the rabbit beta globin polyA signalor the bovine growth hormone polyA signal, as well as polyA signals ofviral origin, such as the SV40 late polyA region. In one embodiment ofthe invention, the vector comprises a chicken beta globinterminator/polyA signal. The multiple cloning site of the vector may belocated between the promoter and the polyA signal. In some embodiments,restriction sites may also be included downstream of the polyA signal.

The vector may contain more than one expression cassette to allow forexpression of multiple recombinant polypeptides from a single vector. Incertain embodiments, the vector comprises 1, 2, 3, 4 or 5 expressioncassettes.

In an exemplary embodiment, the vector is pUHAB. The expression “pUHAB”refers to a vector comprising of the nucleotide sequence of SEQ ID NO: 1(as shown in FIG. 1). The multiple cloning site of the pUHAB vector isflexible enough to incorporate a variety of recombinant genes incomplex, multicistronic arrangements for expression in mammalian cells.Further, two expression cassettes containing the gene of interest andthe TK-Hygromycin drug resistance gene can be transferred together fromthe vector of the invention to a different vector using either Eco571 orBssHII restriction enzymes to generate a new vector containing more thantwo mammalian expression cassettes.

Transfection may result in transiently transfected cell lines, in whichthe vector is maintained episomally and has not integrated into thegenome. Transfection may also result in stably transfected cell lines,in which parts of the vector are stably integrated into the genome ofthe host cell, e.g., by random, non-homologous recombination events. Astable transfection may result in loss of parts of the vector sequencethat are not directly related to expression of the recombinant geneproduct, e.g., bacterial copy number control regions. Accordingly, astably transfected host cell is defined as a host cell that hasintegrated at least part or different parts of the expression vectorinto its genome.

Genes

Any of several genes may be inserted into the plasmids of the presentinvention, for example, immunoglobulins, e.g., which bind specificallyto IGF1R. Plasmids of the present invention encoding any of thefollowing target immunoglobulin amino acid sequences form part of thepresent invention.

See international application publication no. WO2003/100008 which isincorporated herein by reference in its entirety.

2C6 heavy chain (SEQ ID NO: 13) MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEVSGISWNSGSKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIRIGVAASYYFGMDVWGHGTTVTVSS (SEQ ID NO: 14) 2C6 CDR-H1: GFTFDDYAMH (SEQ IDNO: 15) 2C6 CDR-H2: GISWNSGSKGYVDSVKG (SEQ ID NO: 16) 2C6 CDR-H3:DIRIGVAASYYFGMDV 2C6 Light chain (SEQ ID NO: 17) MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQ GTKLEIK (SEQ ID NO:18) 2C6 CDR-L1: RASQGISSVLA (SEQ ID NO: 19) 2C6 CDR-L2: DASSLES (SEQ IDNO: 20) 2C6 CDR-L3: QQFNSYPYT 9H2 Heavy chain (SEQ ID NO: 21)MDWTWRILFLVAAATGAHS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTVYMELSSLRSEDTAVYYCARGGMPVAGPGYFYYYGMDVWGQGTTVTVSS (SEQ ID NO: 22) 9H2 CDR-H1: GYTFTSYVMH (SEQID NO: 23) 9H2 CDR-H2: WINAGNGNTKYSQKFQG (SEQ ID NO: 24) 9H2 CDR-H3:GGMPVAGPGYFYYYGMDV 9H2 Light chain (SEQ ID NO: 25) METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYCCQQYGSSPWTFG QGTKVEIKRT (SEQ IDNO: 26) 9H2 CDR-L1: RASQSVSRSYLA (SEQ ID NO: 27) 9H2 CDR-L2: GASSRAT(SEQ ID NO: 28) 9H2 CDR-L3: QQYGSSPWT Heavy chain immunoglobulinvariable region # 1.0 sequence (SEQ ID NO: 29) E VQLLESGGGL VQPGGSLRLSCTASGFTFSS YAMNWVRQAP GKGLEVIVSAI SGSGGTTFYA DSVKGRFTIS RDNSRTTLYLQMNSLRAEDT AVYYCAKDLG WSDSYYYYYG MDVWGQGTTV TVSS; Light chainimmunoglobulin variable region # 1.0 sequence (SEQ ID NO: 30) DIQMTQFPSSLSASVGDR VTITCRASQG IRNDLGWYQQ KPGKAPKRLI YAASRLHRGV PSRFSGSGSGTEFTLTISSL QPEDFATYYC LQHNSYPCSF GQGTKLEIKR;

Embodiments of the invention include those wherein the plasmid includesmore than one immunoglobulin, for example, a combination of any of thoseset forth herein (e.g., heavy chain lg. #1.0 and light chain lg. #1.0;or LCC and HCA; or LCF and HCA; or LCC and HCB).

Protein Expression and Purification

A further aspect of the present invention relates to a method for theproduction of a recombinant protein (e.g., anti-TGFβ, anti-IGF1R,anti-IL-23, anti-EGFR, anti-IL-17, anti-PD1, anti-IL-1, anti-HGF),comprising the steps of: a) transfecting a host cell or host cell line(e.g., a mammalian host cell or host cell line) with an expressionvector according to the invention; b) culturing the cell underappropriate conditions to enable growth and/or propagation of the celland expression/production of the recombinant protein; and, optionally c)harvesting the recombinant protein produced. Methods for harvesting(isolating and/or purifying) a given protein from, e.g., a cell, a cellculture or the medium in which cells have been cultured are well knownin the art. By way of nonlimiting example, proteins can be isolatedand/or purified from biological material by salt or alcoholprecipitation (e.g., ammonium sulfate precipitation or ethanolprecipitation), affinity chromatography (e.g., used in conjunction witha purification tag); fractionation on immunoaffinity or ion-exchangecolumns; high pressure liquid chromatography (HPLC); reversed-phaseHPLC; chromatography on silica or on a cation-exchange resin such asDEAE; chromatofocusing;); isoelectric focusing; countercurrentdistribution; SDS-PAGE; gel filtration (using, e.g., Sephadex G-75); andprotein A Sepharose columns to remove contaminants such as IgG. Suchpurification methods are well known in the art and are disclosed, e.g.,in “Guide to Protein Purification”, Methods in Enzymology, Vol. 182, M.Deutscher, Ed., 1990, Academic Press, New York, N.Y.

Growth of mammalian cells in liquid aqueous culture is well known in theart. Examples of mammalian cell culture growth media which are known inthe art include EX-CELL ACF, CHO medium (Sigma-Aldrich (St. Louis, Mo.);discussed further below), DMEM, DMEM/F-12, F-10 Nutrient Mixture, RPMIMedium 1640, F-12 Nutrient Mixture, Medium 199, Eagle's MEM, RPMI, 293media, and Iscove's Media.

Cell growth can be performed in any of several systems. For example,cell growth can be done in a simple flask, e.g., a glass shake flask.Other systems include tank bioreactors, bag bioreactors and disposablebioreactors. A tank bioreactor includes, typically, a metal vessel(e.g., a stainless steel jacketed vessel) in which cells are growth in aliquid medium. Tank bioreactors can be used for a wide range of culturevolumes (e.g., 100 l, 150 l, 10000 l, 15000 l). Tank bioreactors oftenhave additional features for controlling cell growth conditions,including means for temperature control, medium agitation, controllingsparge gas concentrations, controlling pH, controlling O₂ concentration,removing samples from the medium, reactor weight indication and control,cleaning hardware, sterilizing the hardware, piping or tubing to deliverall services, adding media, control pH, control solutions, and controlgases, pumping sterile fluids into the growth vessel and, supervisorycontrol and a data acquisition. Classifications of tank bioreactorinclude stirred tank reactors wherein mechanical stirrers (e.g.,impellers) are used to mix the reactor to distribute heat and materials(such as oxygen and substrates). Bubble column reactors are tallreactors which use air alone to mix the contents. Air lift reactors aresimilar to bubble column reactors, but differ by the fact that theycontain a draft tube. The draft tube is typically an inner tube whichimproves circulation and oxygen transfer and equalizes shear forces inthe reactor. In fluidized bed reactors, cells are “immobilized” on smallparticles which move with the fluid. The small particles create a largesurface area for cells to stick to and enable a high rate of transfer ofoxygen and nutrients to the cells. In packed bed reactors cells areimmobilized on large particles. These particles do not move with theliquid. Packed bed reactors are simple to construct and operate but cansuffer from blockages and from poor oxygen transfer. A disposablebioreactor is a disposable, one-time use bioreactor. Often, disposablebioreactors possess features similar to non-disposable bioreactors(e.g., agitation system, sparge, probes, ports, etc.).

Particularly where a polypeptide is isolated from a cellular or tissuesource, it is preferable to include one or more inhibitors ofproteolytic enzymes in the assay system, such as phenylmethanesulfonylfluoride (PMSF), Pefabloc SC, pepstatin, leupeptin, chymostatin andEDTA.

In some embodiments, the protein of interest is with a secondpolypeptide or polynucleotide moiety, which may be referred to as a“tag” or “marker”. A tag may be used, for example, to facilitatepurification or detection of the polypeptide after expression. A fusedpolypeptide may be constructed, for example, by in-frame insertion of apolynucleotide encoding the tag on the 5′ or 3′ end of thepolynucleotide encoding the polypeptide to be expressed. The fusedpolynucleotide may then be expressed in the expression system of theinvention. Such tags include glutathione-S-transferase (GST),hexahistidine (His6) tags, maltose binding protein (MBP) tags,hemagglutinin (HA) tags, cellulose binding protein (CBP) tags and myctags. Detectable tags such as ³²P, ³⁵S, ³H, ^(99m)TC, ¹²³I, ¹¹¹In, ⁶⁸Ga,¹⁸F, ¹²⁵I, ¹³¹I, ^(113m)In, ⁷⁶Br, ⁶⁷Ga, ^(99m)Tc, ¹²³I, ¹¹¹In and ⁶⁸Gamay also be used to label the polypeptides and polynucleotides of theinvention. Methods for constructing and using such fusions are veryconventional and well known in the art.

One skilled in the art appreciates that purification methods suitablefor the polypeptide of interest may require modification to account forchanges in the character of the polypeptide upon expression inrecombinant cell culture.

Kits

The vectors of the invention may be provided in a kit. The kits of theinvention may include, in addition to one or more vectors, any reagentwhich may be employed in the use of the vector. In one embodiment, thekit includes reagents necessary for transformation of the vectors intomammalian cells. For example, the kit may include reagents for a calciumphosphate transformation procedure: calcium chloride, buffer (e.g.,2×HEPES buffered saline), and sterile, distilled water. In anotherembodiment, the kit includes reagents for a DEAE-Dextran transformation:Chloroquine in PBS, DEAE-dextran in PBS and Phosphate buffered saline.In yet another embodiment, reagents for a liposome transformation areincluded in the kit: Liposomes extruded from DOTAP/cholesterol extrudedliposomes. For example, the kit may include the cationic lipid-basedtransfection reagent Lipofectamine™ (Invitrogen Life Technologies;Carlsbad, Calif.).

The kit may include reagents required for bacterial transformation ofthe vectors of the invention. For example, the kit may includetransformation competent bacteria (e.g., DH1, DH5, DH5α, XL1-Blue, SURE,SCS110, OneShot Top 10, or HB101).

The kit may include growth media or reagents required for making growthmedia. For example, in one embodiment, the kit can include fetal calfserum or DMEM (Dulbecco/Vogt modified Eagle's (Harry Eagle) minimalessential medium) for growth of mammalian cells. In another embodiment,the kit can contain powdered Luria broth media or Luria broth platescontaining an appropriate antibiotic (e.g., ampicillin or kanamycin) forgrowing bacteria.

Components supplied in the kit may be provided in appropriate vials orcontainers (e.g., plastic or glass vials). The kit can includeappropriate label directions for storage, and appropriate instructionsfor usage.

EXAMPLE

The following example is provided to further describe the presentinvention and should not be construed as a limitation thereof. The scopeof the present invention includes any and all of the methods which areset forth below in the following example.

Example 1 Construction of pUHAB

The multiple cloning site of pUHAB, as shown in SEQ ID NO: 2, wassynthesized using PCR. The PCR product and the vector of SEQ ID NO: 3(Vector A; see FIG. 3) were digested by the restriction enzymes AflIIand BamHI. The digested PCR product was then ligated to the digestedVector A to form the pUHAB vector (SEQ ID NO: 1). pUHAB was transformedinto cells, which were positively selected for presence of the vector.Finally, the integrity of the pUHAB multiple cloning site was confirmedby sequencing.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A plasmid vector comprising a multiple cloning site comprising atleast twelve of the following restriction sites: AflII, HpaI, AvrII,EcoRV, Acc651, KpnI, PacI, NotI, BstZ17I, SrfI, ApaI, NheI, BglII, SphI,and BamHI.
 2. The vector of claim 1, wherein said vector comprises allof said restriction sites
 3. The vector of claim 2, wherein said vectorcomprises the following multiple cloning site:5′-AflII-HpaI-AvrII-EcoRV-Acc651-Kpn1-PacI-NotI-BstZ17I-SrfI-ApaI-NheI-BglII-SphI-BamHI-3′.4. The vector of claim 3, wherein the multiple cloning site comprisesthe nucleotide sequence set forth in SEQ ID NO:
 2. 5. The vector ofclaim 1, comprising a promoter located upstream of or within themultiple cloning site.
 6. The vector of claim 5, wherein the promoter isthe human cytomegalovirus (hCMV) promoter.
 7. The vector of claim 5,wherein said promoter is operably associated with an intron thatenhances expression from said promoter.
 8. The vector of claim 7,wherein the nucleotide sequence of said intron comprises the nucleotidesequence of SEQ ID NO:
 4. 9. The vector of claim 1, further comprisingat least three elements selected from the group consisting of: (a) aselectable marker for eukaryotic cells; (b) a prokaryotic origin ofreplication; (c) a bacterial drug resistance marker; and (d) a phagef1(+) region.
 10. The vector of claim 9, wherein said vector comprisesall of said elements.
 11. The vector of claim 9, wherein said selectablemarker for eukaryotic cells is a hygromycin (TK-Hygromycin) drugresistance gene operably linked to a thymidine kinase promoter.
 12. Thevector of claim 9, wherein said prokaryotic origin of replication is theColE1 origin of replication.
 13. The vector of claim 9, wherein saidbacterial drug resistance marker is a kanamycin resistance gene.
 14. Thevector of claim 1 comprising a terminator/polyA addition site.
 15. Acomposition comprising the vector of claim 1 and an isolated bacterialcell or mammalian cell.
 16. The vector of claim 1, comprising thenucleotide sequence set forth in SEQ ID NO:
 1. 17. The vector of claim1, comprising a heterologous DNA sequence encoding a polypeptide, whichDNA sequence is operably linked to a promoter and an intron.
 18. Thevector of claim 17 wherein the polypeptide is an antibody immunoglobulinchain.
 19. The vector of claim 18 wherein the antibody bindsspecifically to IGF1R.
 20. The vector of claim 19 wherein theimmunoglobulin chain comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 5-30.
 21. An isolated plasmid vectorcharacterized by the plasmid map of FIG. 1 or FIG.
 22. An isolatedplasmid vector comprising the nucleotide sequence set forth in SEQ IDNO:
 3. 23. An isolated host cell comprising the vector of claim
 1. 24. Ahost cell line comprising the host cell of claim
 23. 25. A method forproducing a recombinant polypeptide in an isolated host cell, comprisingintroducing the vector of claim 17 into the host cell under conditionswhich allow for expression of the polypeptide.
 26. The method of claim25, further comprising purifying the polypeptide.
 27. The method ofclaim 25 wherein the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5-30.
 28. A kitcomprising the vector of claim 1 and instructions for use of the vector.